The proposed research will consist of biophysical studies that probe the interactions between the HIV-1 regulatory protein, Rev, and the viral RNA structure, the Rev Response Element (RRE). The goal of the proposed research is to elucidate the kinetic pathway of Rev, which activates nuclear export of unspliced and partially spliced RNAs, and thus plays an essential role in HIV-1 replication. Novel single-molecule fluorescence imaging techniques will be developed and utilized to dissect the assembly and disassembly pathways of the Rev-RRE complex, and to illuminate the role of various host cell factors in assembly and dissociation. Specifically, total internal reflection fluorescence microscopy and two-color colocalization imaging of fluorescently-labeled Rev monomers and host factors will be employed to achieve the stated goals. Due to the complex kinetic pathway of Rev, including features such as Rev oligomerization, Rev- RRE complex formation and dissociation, and direct and mediated interactions with host cell factors, traditional bulk biochemical techniques have failed to provide a detailed picture of the Rev and Rev-RRE kinetic pathways. Therefore, while Rev is a promising therapeutic target, due to its essential role in HIV-1 replication, effective Rev-based therapies have yet to be developed. This research will directly probe the interactions between Rev, the RRE, and various host factors on a single-molecule level, and thus will be able to define detailed kinetic pathway(s) associated with each step in the Rev cycle, as well as determine the stoichiometries of Rev, RRE, and host factors at each step. Consequently, this research will answer many unsolved questions regarding the Rev kinetic pathway and be able to advance progress towards the implementation of HIV-1 therapies that target Rev. PUBLIC HEALTH RELEVANCE: The HIV-1 regulatory protein, Rev, is essential to HIV-1 replication, and thus it is a promising therapeutic target in the fight against the worldwide AIDS epidemic. However, due to the complex kinetic pathway of Rev, therapies that target Rev have yet to be actualized. This research will elucidate many details about Rev by directly probing the interactions between Rev, RNA and various host cell proteins on a single- molecule level, and thus will be able to advance the development of Rev-based HIV-1 therapies.